The goal of this proposal is to develop a sensitive, high-throughput localized surface plasmon resonance assay for the detection of a wide range of biomarkers at levels reaching down to that of single molecules and with a highly scalable dynamic range. We have recently shown that plasmon resonant nanoparticles are highly sensitive to their proximity to nearby conductive substrates. They display large spectral shifts in resonance (or color) as well as changes in scattered light polarization upon nanometer-scale changes in distance from a nearby metal film. In addition, such nanoparticles are also effective labels for biomolecules because of their ease of functionalization and detection. Proposed here is a scheme whereby plasmon resonant nanoparticles and metal surfaces are functionalized with capture molecules designed to recognize specific biomarkers varying in size from small molecules/DNA targets to proteins. Upon exposure to clinical samples, the functionalized nanoparticles bind target analytes and are then directed to specific areas on conductive substrates defined by surface patterns of complimentary capture molecules. The completely novel innovation proposed here is that targets bound to nanoparticles will be detected and discriminated from nonspecific adsorption of nanoparticles without targets by searching in a digital image for a predetermined nanoparticle color and/or scattering polarization that is specific to the physical size (and hence, spacing between nanoparticle and film) determined by bound target. Specific aims of this proposal are as follows: 1. Optimization of the sensor response. 2. Design of "receptor" functionalized NPs and fabrication of sensor chips. 3. Detect clinical analytes. Public Health Relevance: As our knowledge of disease expands the need to reliably detect biomolecules of various sizes and concentrations becomes more important. The goal of this work is to develop a novel detection platform that is scalable from the single molecule level to the multiplexed assay level where signal is easily disguishable from background noise, thus increasing the accuracy, sensitivy, and applicability of clinical biomarker assays.